The present invention relates to methods of identifying or screening for drugs or agents useful in treatment of psychiatric or neurological disorders, and relates in particular to screening for non-steroidal drugs or agents.
The present invention is based, in part, on experimental work, some of which is reported in the following articles, the disclosures of which are incorporated herein by reference:
(a) Cheney, D. L., Uzunov, D., Costa, E., Guidotti, A. (1995a); Gas chromatographic mass fragmentographic quantitation of 3xcex1-hydroxy-5xcex1-pregnan-20-one (allopregnanolone) and its precursors in blood and brain of adrenalectomized and castrated rats. J. Neurosei, 15:4641-4650.
(b) Cheney, D. L., Uzunov, D., Guidotti, A. (1995b): Pregnanolone sulfate antagonizes dizocilpine amnesia: Role for allopregnanolone. NeuroReport 6:1697-1700.
(c) Guidotti, A., Uzunov, D., Auta, J., Costa, E. (1996): Application of gas chromatography mass fragmentography with negative ion chemical ionization technology to measure neurosteroids and their biosynthesis rate in the rat brain In: Genazzani, A. R., Petraglia, F., Purdy, R. H., editors. The Brain: Source and Target for Sex Steroid Hormones. New York:Parthenon, pp 24-41.
(d) Uzunov, D. P., Cooper, T. B., Costa, E., Guidotti, A. (1996): Fluoxetine-elicited changes in brain neurosteroid content measured by negative ion mass fragmentography. Proc. Natl. Acad. Sci. USA 93:12599-12604.
(e) Uzunova, V., Sheline, Y., Davis, J. M., Rasmusson, A., Uzunov, D. P., Costa, E., Guidotti, A. (1998): Increase in cerebrospinal fluid content of neurosteroids in patients with unipolar major depression who are receiving fluoxetine or fluvoxamine. Proc. Natl. Acad. Sci. USA 95: 3239-3244.
(f) Matsumoto, K., Uzunova, V., Pinna, G., Taki, K., Uzunov, D. P., Watanabe, H., Mienville, J. M., Guidotti, A., Costa, E. (1999): Permissive role of brain allopregnanolone content in the regulation of pentobarbital-induced righting reflex loss. Neuropharmacol, 38(7):955-964.
It is becoming increasingly clear that naturally-occurring steroidal modulators of neuronal function do exist in the brain acting as very potent endogenous regulators of the function of the brain""s most abundant inhibitory (y-aminobutyric acid) or excitatory (glutamatergic) neurotransmitter systems. The central nervous system (CNS) has the capacity to synthesize such steroids de novo from cholesterol independently of peripheral hormonal sources. These steroids are referred to herein as xe2x80x9cneurosteroids.xe2x80x9d Neurosteroids that are pharmacologically potent modulators of neuronal activity are defined herein as neuroactive steroids. The pharmacological response induced by some neurosteroids is similar to that of the benzodiazepines (Valium-like drugs), and in non-technical language are likely to be the brain""s own naturally occurring Valium-like compounds. Specifically, the neurosteroid 5xcex1-pregnan-3xcex1-ol-20-one (allopregnanolone, ALLO) at low nanomolar concentrations positively modulates xcex3-aminobutyric acid type A (GABAA) receptor function, rapidly affecting brain excitability.
An example of biosynthesis of neurosteroids from cholesterol is illustrated in FIG. 1, which shows the major metabolic pathway for neurosteroid biosynthesis in the CNS. Pregnenolone (PREG) is formed from cholesterol by the cytochrome P45O side chain cleavage (P450SCC) enzyme. PREG is transformed into progesterone (PROG) by 3xcex2-hydroxysteroid dehydrogenase isomerase (3xcex2-HSD) and PROG is transformed into 5xcex1-dihydroprogesterone (5xcex1-DHP) by 5xcex1-reductase. Finally, ALLO can be synthesized from 5xcex1-dihydroprogesterone. (5xcex1-DHP) by the action of the 3xcex1-hydroxysteroid oxidoreductase (3xcex1-HSOR) enzyme. This enzyme has two reversible functionsxe2x80x94oxidative and reductive. The reduction of 5xcex1-DHP to ALLO is dependent upon the availability of the cofactors NADPH or NADH, whereas the opposite step, which catalyzes the oxidation of ALLO to 5xcex1-DHP is NAD/NADP dependent. Currently, the major pathway of ALLO metabolism and deactivation in the brain is thought to be the degradation of ALLO to 5xcex1-DHP by the oxidative action of the 3xcex1-HSOR. We have demonstrated that ALLO is unevenly distributed in brains of adrenalectomized and castrated (ADX/CX) rats, suggesting the existence of local regulatory mechanisms for its production and metabolism.
Referring to FIG. 2, in addition to the conversion to 5xcex1-DHP by the 3xcex1-HSOR, an alternative route for the deactivation of allopregnanolone is through the metabolism to 20xcex1-hydroxyallopregnanolone (5xcex1-pregnan-3xcex1,20xcex1-diol)(20xcex1-hydroxy ALLO) by the action of the 20xcex1-hydroxysteroid dehydrogenase (20xcex1-HSD). The 20xcex1-HSD also metabolizes progesterone to 20xcex1-hydroxyprogesterone (4-pregnen-20xcex1-hydroxy-3-one)(20xcex1-hydroxy PROG), thus decreasing the endogenous pool of progesterone available to be metabolized to 5xcex1-DHP and ultimately to allopregnanolone. Since both actions of the 20xcex1-HSD would result in a significant decrease in the CNS levels of allopregnanolone, non-steroidal compounds that selectively inhibit the 20xcex1-HSD would prevent the 20xcex1-HSD-mediated depletion of the neuroactive steroid allopregnanolone in the brain.
Measurements of allopregnanolone in brain microdialysates obtained from freely moving rats have revealed that allopregnanolone is present in the extracellular compartment. These findings strongly suggest that allopregnanolone is released by glial cells and neurons and it can accumulate in the synaptic cleft in concentrations sufficient to activate the GABAA receptor. It remains to be understood whether an active reuptake mechanism involved in maintaining a physiological synaptic concentration of allopregnanolone exists. If such a mechanism does exist, then potential inhibitors of the reuptake of allopregnanolone would increase its accumulation in the vicinity of its target receptor.
Several lines of evidence indicate that a potentiation of serotonergic neurotransmission underlies the therapeutic response to various types of antidepressants. Fluoxetine and other selective serotonin reuptake inhibitors (SSRIs) have a spectrum of clinical efficacy that is different, greater, and quite often superior to that of other antidepressants. Hence, it is becoming increasingly clear that the ability of SSRIs to enhance serotonin activity is not the only mechanism responsible for the large spectrum of favorable clinical actions of the SSRIs.
Furthermore, the etiologies of premenstrual dysphoric disorder (PMDD) and premenstrual syndrome (PMS) are of considerable relevance to neurosteroids as their symptoms (anxiety, mood fluctuations, susceptibility to seizures, etc.) are associated with a precipitous decline in circulating levels of progesterone and its GABAA receptor active metabolite ALLO. In a recent study, it has been concluded that subjects with PMS manifest significantly lower levels of serum ALLO in the luteal phase when compared to controls. Interestingly, fluoxetine has been shown to be effective in the treatment of premenstrual dysphoria, displaying a much faster therapeutic response than the usual lag of three to six weeks required for SSRIs to become effective in alleviating symptoms of depression, suggestive that an enhancement of serotonergic neurotransmission is not the only mechanism underlying, the therapeutic action of fluoxetine in premenstrual dysphoria.
However, substantial difficulties prevent a direct therapeutic intervention with neurosteroids. For example, the results of the systemic administration of the 3xcex1,5xcex1-reduced derivatives of PROG, acting as positive allosteric modulators of GABA action at GABAA receptors, indicate that the doses required to elicit a clear anxiolytic, antidysphoric, and antiepileptic activity may also produce profound sedation, motor impairment, or ataxia. Furthermore, the endogenous neurosteroids have a very short metabolic half-life and thus a limited bioavailability. All these properties compromise the development of these naturally occurring compounds as therapeutic agents.
In view of the foregoing, several drug companies are producing synthetic analogs of neurosteroids in an attempt to develop new drugs for the treatment of neuropsychiatric disorders. For example, CoCensys, Inc. has created a variety of synthetic analogs of endogenous neuroactive steroids, called epalons, that are being clinically evaluated for anesthesia and various neurological and psychiatric disorders. These compounds, however, although possessing less metabolic liability than endogenous neurosteroids, are still highly lipophilic, giving rise to serious formulation problems and consequently to decreased CNS bioavailability, ultimately having compromised the development of these compounds as neuropsychiatric therapeutic agents.
Our approach to utilizing the therapeutic potential of neuroactive steroids is conceptually different. Judging from our accumulated experimental data we postulate that neurosteroids serve a physiological role as endogenous modulators of brain function. Moreover, we have clearly demonstrated the existence of a link between a deficiency in endogenous neurosteroid bioavailability and symptoms of depression. The screening technology that we have invented is unique in that it will aid the discovery and development of non-steroidal neuropsychiatric drugs that would regulate the endogenous levels of neuroactive steroids, as opposed to the prior approach of an exogenous administration of neurosteroids or synthetic neurosteroid-like compounds. Our screening technology is designed to identify compounds which would (a) selectively affect some rate-limiting steps of the biosynthesis of the endogenous neuroactive steroids in the brain, such as the conversion of 5xcex1-DHP to ALLO and/or vice versa, the degradation of ALLO to 5xcex1-DHP by the 3xcex1-HSORs (see FIG. 1), or (b) would inhibit the reuptake of the endogenous neuroactive steroids by neurons or glial cells. These compounds are collectively termed Selective Neurosteroid Regulating Agents (SNRAs).
It is a general object of the invention to provide an improved approach to identifying neuropsychiatric drugs which avoids disadvantages of prior techniques and affords other advantages.
An important feature of the invention is the provision of a method of screening for non-steroidal neuropsychiatric agents based upon the ability of the agent to affect the bioavailability of endogenous neuroactive steroids.
In connection with the foregoing feature, another feature of the invention is the provision of a method of the type set forth, which involves determining the ability of the agent to selectively regulate a rate-limiting step in the bio-control of the bioavailable amount of an endogenous neuroactive steroid.
A still further feature of the invention is the provision of a method of the type set forth, wherein the rate-limiting step is a step in biosynthesis of the endogenous neuroactive steroid and/or biodegradation thereof.
Certain ones of these and other features of the invention may be attained by providing a method of screening for non-steroidal neuropsychiatric agents comprising: determining the ability of a candidate non-steroidal agent to selectively regulate or alter the central nervous system content and/or bioavailability of an endogenous neuroactive steroid.
Still further features of the invention are attained by providing a method of the type set forth which includes administering a candidate agent to a warm-blooded mammal and observing the effects of the agent in altering the bioavailability of an endogenous neuroactive steroid in the mammal.
Still other features of the invention are attained by providing a method of the type set forth, which involves administering a candidate agent to a primary culture of rodent cerebellar neurons and/or astrocytes or a cell line culture or brain slices and observing the effect in altering the amount of endogenous neuroactive steroids in the culture or brain slices.